Article Genetic association between AZF region polymorphism and Klinefelter syndrome

RBMOnline - Vol 19. No 4. 2009 547 551 Reproductive BioMedicine Online; www.rbmonline.com/article/3741 on web 21 August 2009 Article Genetic association between AZF region polymorphism and Klinefelter syndrome Dr Lobna Hadjkacem-Loukil obtained M.Sc. and Ph.D. degrees in biology from the University of Sfax, Tunisia, researching into the genetic background of male infertility in the Tunisian population. Her major research interest now is in the human Y chromosome. In 2004 she started molecular analysis of candidate genes involved in infertility on the Y chromosome at the Laboratory of Human Molecular Genetics, Medicine Faculty, University of Sfax. Dr Lobna Hadjkacem-Loukil Lobna Hadjkacem-Loukil 1,4, Myriam Ghorbel 1, Ali Bahloul 2, Hammadi Ayadi 3, Leila Ammar-Keskes 1 1 Laboratory of Human Molecular Genetics, Faculty of Medicine, Sfax 3029, Tunisia; 2 Unit of Research US 17, CHU Hospital Habib Bourguiba, Sfax 3029, Tunisia; 3 Unité Cibles pour le Diagnostic et la Thérapie, Centre de Biotechnologie de Sfax, Tunisia 4 Correspondence: e-mail: lobna_h@lycos.com Abstract Because of conflicting results about the association between azoospermic patients with Klinefelter syndrome (KFS) and azoospermia factor (AZF) polymorphism, and because nothing is known about the association of KFS with partial AZFc deletions, an association study was performed in Tunisian KFS patients. A total of 29 azoospermic patients and 13 fertile men were enrolled in this study. The classical microdeletions were found in six out of nine KFS patients (67%). Gr/Gr deletions and b2/b3 deletions are partial AZFc deletions. One KFS patient without classical microdeletions had a gr/gr deletion. This deletion (gr/gr) was observed in four out of 18 azoospermic patients without chromosomal abnormalities. In addition, two b2/b3 and one AZFc deletion were identified in this group. All KFS patients had elevated plasma FSH and LH concentrations, but normal plasma testosterone concentration. The testis biopsy of three samples with Y microdeletions revealed Sertoli cell-only syndrome. No Y microdeletions or partial AZFc deletions were found in the fertile group. It is concluded that in the patient population KFS patients may harbour Y microdeletions, and screening for these should be part of the diagnostic work-up, particularly in those considering assisted reproductive techniques. However, partial AZFc deletions might not play a role in predisposing genetic background for the phenotype of azoospermic KFS subjects. Keywords: association, AZF polymorphism, azoospermia, Klinefelter syndrome, partial deletions, Y chromosome Introduction In the last few years, considerable progress has been made in understanding the pathogenesis of spermatogenic arrest and infertility. Infertility is often due to hypogonadism; Klinefelter s syndrome (KFS) is the most common cause of hypogonadism and infertility (Tateno et al., 1999). KFS is the most common sex chromosomal abnormality in men, with an incidence of approximately 1 in 500 newborn phenotypic males (Tateno et al., 1999). Men with KFS have a 47,XXY chromosome complement, 15% of whom are classified as mosaic (KFM) with a 46,XY/47,XXY chromosomal complement. KFS is characterized by seminiferous tubular dysgenesis, azoospermia and elevated concentrations of serum gonadotrophins. Studies have shown that the deletion of azoospermia factor (AZF) loci on the long arm of the Y chromosome results in spermatogenic failure and these loci are recurrently deleted in infertile males (Van der Ven et al., 1997; Foresta et al., 1998; Dada et al., 2002; Thangaraj et al., 2003; Chiang et al., 2004; HadjKacem et al., 2006). Previously, men with non-obstructive azoospermia had no therapeutic fertility options except anonymous donor insemination or adoption. The ability to extract spermatozoa from the testes of some men with non-obstructive azoospermia using the multi-biopsy technique of testicular sperm extraction followed by intracytoplasmic sperm injection (ICSI) offers an efficacious therapeutic approach (Schlegel et al., 1997). The possibility of using ICSI with testicular spermatozoa has also been proposed in KFS patients (Denschlag et al., 2004). The widespread use of such procedures has raised 547 Ó 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

548 some interesting and important genetic issues about the inheritance of genetic anomalies, such as Y chromosome microdeletions in the offspring (Cram et al., 2000). AZFc is the most commonly deleted interval in men with azoospermia or severe oligozoospermia (Kuroda-Kawaguchi et al., 2001; Skaletsky et al., 2003). AZFc deletions are generated by intrachromosomal homologous recombination between repeated sequence blocks called amplicons organized in palindromic structures with nearly identical sequences in each palindrome arm. Within the AZFc region there are several candidate fertility genes, including three copies of BPY2 (basic protein on Y chromosome 2), two copies of CDY1 (CDY1a and CDY1b; chromodomain protein, Y chromosome 1), and four copies of the DAZ (deleted in azoospermia) gene family (McElreavey et al., 2000; Kuroda-Kawaguchi et al., 2001). A partial deletion termed gr/gr has been recently described in the AZFc region, and considered as a risk factor for spermatogenic failure in some studies (Repping et al., 2003; Ferlin et al., 2005; Giachini et al., 2005). Other studies suggest that it is a polymorphic deletion with no clinical relevance (Machev et al., 2004). This deletion removes half the AZFc gene content, including two copies of the major AZFc candidate gene, DAZ (Machev et al., 2004). Another deletion termed b2/b3 (1.8 Mb), which also results in the absence of half the AZFc gene compliment, seems to have no effect on fertility status; it is found on a certain chromosome background commonly present in northern Eurasian populations (Machev et al., 2004; Repping et al., 2004). There are conflicting reports on the occurrence of Y chromosome microdeletions in KFS patients (Tateno et al., 1999; Ambasudhan et al., 2003; Choe et al., 2007). Tateno et al. (1999) failed to find microdeletions of the DAZ or the Y chromosome ribonucleic acid recognition motif (YRRM) genes in KFS patients. On the other hand, others have reported a low incidence of Y chromosome microdeletions in oligospermic or azoospermic men (Oliva et al., 1998; Oates et al., 2002; Peterlin et al., 2002; Mitra et al., 2006). Because little information is available on AZF region polymorphism in KFS patients, this study has analysed for the first time the occurrence of partial AZFc deletions and Y chromosome microdeletions in KFS patients. Materials and methods Subjects The study population consisted of a group of 29 nonobstructive azoospermic patients and 13 fertile men. All subjects and controls were of Tunisian ethnic origin. The azoospermic patients were subject to detailed clinical and biological investigations, including cytogenetic and endocrinology studies, physical examination and, when possible, histology of a testis biopsy. The karyotype analysis revealed six cases with a 47,XXY chromosomal pattern (KFS), three KFM cases, one case with 46,XYinv (12) (p12q12), one case with 46,XY+marq and 18 azoospermic patients without chromosomal abnormalities. Semen analysis was done according to World Health Organization (WHO) guidelines (WHO, 1999). The plasma values of FSH, LH and testosterone were determined. Molecular analysis Genomic DNA was extracted from peripheral blood lymphocytes using standard techniques, and amplified by multiplex polymerase chain reaction (PCR). The concentration of the isolated genomic DNA was determined by spectrophotometric analysis at 260 nm. Microdeletion analysis of the Y chromosome Yq AZF region involved two steps. In the first step, aimed at detecting AZFa, AZFb and AZFc microdeletions, multiplex PCR systems were carried out according to the conditions described by Hadjkacem-Loukil et al. (2007). The sex-determining region of Y [SRY, i.e. sequence tagged site (STS) sy14] was included as the internal control. DNA from fertile male subjects was taken as the positive control and DNA from a female subject as the negative control in each reaction. In the second step, two multiplex systems were used, one including the STS primers sy1291, sy1191 and sy1161 and the other including sy1201 and sy1206, to screen the partial AZFc deletions, as described previously (Hucklenbroich et al., 2005). The samples in which the deletions were detected by the above multiplex systems were confirmed by repeating the single primer (simplex) reactions in the presence of both the internal control (SRY) and the positive control. Results The mean age of the samples was 31 years (range 27 40) for normal subjects and 31 years (range 26 43) for azoospermic patients. All cases presented with primary infertility. All patients showed the absence of spermatozoa in the semen. FSH concentrations in the KFS cases were found to be very high [mean: 33.25 ± 1.55 miu/ml; range (20.9 70.1)]. LH concentrations were also found to be high [mean: 14.64 ± 0.85 miu/ml; range (9.3 29.2)]. However, testosterone concentrations were normal [mean: 2.27 ± 0.31 ng/ll; range (0.5 4.29)]. Six out of the 9 (67%) KFS patients showed deletions in AZFa, AZFb and AZFc regions (Table 1). Deletions of STS differ from one patient to another. The extent of the deletions is shown schematically in Figure 1. Three patients (P107, P108 and P61) had deletions in the three regions (AZFa, AZFb and AZFc) (Figure 1). The others (P13, P50 and P101) have deletions in AZFa, either AZFb or AZFc (Figure 1). In comparison, using similar conditions of PCR, only one out of 18 azoospermic patients without chromosomal abnormalities showed a deletion in the AZFc region and none of the fertile men had any Y chromosome microdeletions. Testicular biopsy was carried out in eight patients with KFS. Two cases with 47,XXY and AZFa, AZFb and AZFc deletions had Sertoli cell-only syndrome (SCO) (Table 2). One case with 47,XXY/ 46,XY and AZFa, AZFb and AZFc deletions had SCO syndrome. One case with 47,XXY and AZFa deletions had bilateral hypotrophy. Two cases with 47,XXY/ 46,XY and AZFb or AZFc deletions had bilateral hypotrophy.

Table 1. Y chromosome microdeletions and partial azoospermia factor c (AZFc) deletions data. Y chromosome microdeletion AZFa AZFb AZFc AZFa + b + c Total Partial AZFc deletion gr/ gr b2/ b3 AZFc Azoospermia with KFS 1 1 1 3 6 1 0 0 1 (n =9) Azoospermia without CA 0 0 1 0 1 4 2 1 7 (n = 18) Fertile (n = 13) 0 0 0 0 0 0 0 0 0 CA = chromosomal abnormalities; KFS = Klinefelter syndrome. Total Figure 1. A schematic representation of the human Y chromosome showing the extent of deletions in all Klinefelter syndrome patients. Table 2. Y chromosome microdeletions and clinical parameters. Karyotype STS deletion Clinical parameters P106 47,XXY No deletions Bilateral atrophy P111 47,XXY No deletions SCO P118 47,XXY No deletions ND P107 47,XXY AZFa, AZFb and AZFc SCO P61 47,XXY/46,XY AZFa, AZFb and AZFc SCO P108 47,XXY AZFa, AZFb and AZFc SCO P13 47,XXY AZFa Bilateral hypotrophy P50 47,XXY/46,XY AZFc Bilateral hypotrophy P101 47,XXY/46,XY AZFb Bilateral hypotrophy AZF = azoospermia factor; ND = not determined; SCO = Sertoli cell only syndrome; STS = sequence tagged site. Only one KFS patient without AZFa, AZFb and AZFc microdeletions had a gr/gr deletion. Three different patterns of partial deletions within the AZFc region were observed in seven men in the azoospermic group without chromosomal abnormalities. Four (22%) had gr/gr deletions, two (11%) had a b2/b3 deletions and one (5%) had AZFc deletion pattern (Table 2). No partial deletions were found in the fertile group. Discussion This study is a detailed study on Y chromosome microdeletions in KFS patients with 14 sets of primers. There are conflicting reports on the occurrence of Y chromosome microdeletions in KFS patients (Tateno et al., 1999; Ambasudhan et al., 2003; Choe et al., 2007). Tateno et al. (1999) failed to find microdeletions of DAZ or YRRM genes in KFS patients. On the other hand, others have reported a low incidence of Y microdeletions in oligospermic or azoospermic men (Oliva et al., 1998; Oates et al., 2002; Peterlin et al., 2002; Mitra et al., 2006). Nevertheless, the current study illustrates deletions in the AZFa, AZFb and AZFc regions, in contrast to the deletions in the AZFc region observed in KFS and oligo- or azoospermic patients by others (Oliva et al., 1998; Oates et al., 2002; Peterlin et al., 2002) and AZFa and AZFb in KFS patients by Mitra et al. (2006). The AZFa, AZFb and AZFc combined deletions were found in 33.3% of patients. The higher percentage of deletions observed in this population could be explained by the Tunisian origin. In parallel studies using the same sets of PCR primers, infertile men (n = 210) showed an incidence of Y chromosome microdeletions in 48% (Hadjkacem-Loukil et al., 2007). However, using the recommendations of the European Academy of Andrology 549

550 guidelines (Simoni et al., 1999), an overall deletion frequency of 16% was found (HadjKacem et al., 2006). Several partial AZFc deletions have been described in earlier studies (Ferlin et al., 2005). These deletions result in the absence of several AZFc genes and in the case of the gr/gr deletion, it has been suggested to be an important genetic risk factor for spermatogenic failure (Repping et al., 2003; Ferlin et al., 2005). This study analysed for the first time the occurrence of partial deletions of the AZFc region in a small sample of Tunisian KFS patients. Using a PCR approach, these types of partial deletions, gr/gr deletion, b2/b3 deletion and AZFc deletion, were found in the present study population. The gr/gr deletions were present in both KFS patients (11%) and azoospermic patients without chromosomal abnormalities (22%); these deletions in were not found in the control group. This result is similar to recent data reporting a significant correlation between the presence of gr/gr deletion and spermatogenic failure in Spanish men (de Llanos et al., 2005). The b2/b3 deletion was found in only two azoospermic patients without chromosomal abnormalities (2/18); the AZFc deletion was found in only one azoospermic patient without chromosomal abnormalities. In this study, there is a strong association between classical AZF deletions and Klinefelter syndrome, but no evidence of an association between phenotype and genotype in the case of gr/gr and b2/b3 deletions in the present population. Although the nature of the histological changes at the testicular level still needs to be defined, patients with KFS are known to have progressive deterioration of their testicular architecture. The observed high concentrations of FSH in the present study are in agreement with very low concentrations of inhibin-b and high concentrations of FSH reported in KFS patients (Tomasi et al., 2003). The testis biopsy report from eight patients showed an SCO type of morphology in four cases and bilateral hypotrophy in others. The findings of Kamp et al. (2001) suggest a high frequency of AZFa deletions in men with SCO syndrome. It can also be interpreted that deletions in AZFa give rise to more severe phenotypes such as SCO syndrome which contrasts with the present results. For SCO patients, deletions were found in the three regions. In conclusion, due to observed Y chromosome microdeletions in KFS patients, the analysis may be imperative in routine clinical follow-up of such cases, followed by genetic counselling with respect to the risk of transmitting Y chromosome microdeletions to the male progeny, if the patients opt for assisted reproductive techniques. 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